Wax-embedded tissue samples are convenient reservoirs of nucleic acid information. The presence of mRNA in such samples can provide extremely useful information about the expression of genes in certain cell types that have been preserved in this manner. However, when efforts have been made to obtain information about the mRNA in such samples, it is clear that extensive degradation of the cellular RNA exists.
Some researchers have reported detection of mRNA in such preserved tissues by in situ hybridization. Efforts have been made to extract mRNA from such samples and individual species of certain mRNA have been detected by PCR, however, these efforts are generally limited because the mRNA is degraded to small fragments usually less than 200 or 250 nucleotides in length. Because of the extent of degradation of the mRNA, efforts to attempt to obtain a representation of all species of mRNA from such samples have been deemed to be futile.
Further, about 95% of cellular RNA is ribosomal RNA (rRNA). Separation of the mRNA from the rRNA is difficult in samples such as these. The rRNA needs to be eliminated in gene expression studies because it does not reflect expression of the cellular genes. Common methods for separating mRNA from rRNA, such as the oligo (dT) method, operate by selecting for polyadenylated RNA. These methods are not very effective in these types of preserved cell samples. They automatically exclude much of the mRNA because the degradation which has occurred in the sample results in a high percentage of mRNA that does not have a polyA tail.
Also, when RNA is being studied, DNA must be removed to prevent background problems that result from its presence. Though RNA is typically separated from DNA by precipitating it away from the DNA, this method has the disadvantage that the RNA fragments which are obtained are of the same or similar size thus, limiting the representation of the total RNA population. Since paraffin-embedded tissues are a relatively convenient source of a variety of tissue types, it would be highly desirable to develop a method of obtaining a complete representation of the cellular mRNA from paraffin-embedded tissue samples for genomic analysis.